Post-translational acetylation of the ε-amino groups of specific lysine side-chains in the N-terminal domains (N tails) of the core histones H2A, H2B, H3 and H4 in the chromatin of eukaryotic cells is a central mechanism for regulating chromatin structure and function. The N tails, which protrude from the nucleosome, the fundamental unit of the eukaryotic chromosome, are conformationally variable. They alternate between helical and unstructured segments according to their ionic environment, specifically polyamines, as well as inorganic cations, (Baneres, J. L., Martin, A., and Parello, J. The N tails of histones H3 and H4 adopt a highly structured conformation in the nucleosome. J Mol Biol, 273: 503-508, 1997) and unpublished results, and are capable of forming regulatable contacts with DNA and proteins. By eliminating the positive charges of the lysine side chains, acetylation reduces the interaction of the N tails with DNA, increases DNA accessibility, and promotes the recruitment of transcription factors, DNA polymerases, and DNA repair proteins to chromatin.
The level of acetylation is the result of opposing activities of two families of enzymes, namely the histone acetyl transferases (HATs), which transfer an acetyl group from acetyl coenzyme A (CoA) to the lysine side-chain, and the histone deacetylases (HDACs), which catalyze amide hydrolysis with the release of the acetyl group. These enzymes are now known to interact directly with transcriptional regulators, adding support to the earlier studies linking acetylation to transcription. More recently, histone acetylation has been implicated in the processes of DNA repair, histone deposition after DNA synthesis, and replication fork initiation, and therefore has broad relevance to most chromatin-based activities.
Because the HAT enzymes regulate chromatin structure and function, they are important targets for the development of anti-cancer drugs, but progress in this area has been slow. The first HAT inhibitor to be reported was a multisubstrate adduct, spermidine-CO—CH2—CoA (i.e., Spd-CoA), formed by covalently joining spermidine (Spd) to the S atom of coenzyme A through an acetic acid linkage (Cullis et al. 1982, J Biol Chem 257:12165-9). Spd-CoA was demonstrated to act as an inhibitor of histone and polyamine acetylation under in vitro conditions.
Two isomeric forms of Spd-CoA, i.e., Spd(N1)-CoA and Spd(N8)-CoA, considering the primary amino groups at the N1 or N8 atom of Spd attached to the CoA moiety (inhibitors of the series 1a and 1b, respectively), were subsequently synthesized through a totally regioselective approach and shown to act as HAT inhibitors under in vitro conditions (Parello et al. 1990, C.R.Acad.Sci.Paris, Serie II, 310:1441-1446; Roblot et al., 1993, Tetrahedron 49:6381-6398). This synthetic work opened the path to the synthesis of Spd-CoA analogs in which the coenzyme A was truncated (at the level of the beta-alanine unit, to give series 2a/2b; at the level of the cysteamine unit to give series 3a/3b; we will denote these as Spd-2a/2b and Spd-3a/3b both series in which the polyamine moiety is spermidine or Spd). Similar inhibitors based on a lysine-CoA linkage have been described. Several natural products have also been found to inhibit p300 or PCAF histone acetyltransferase activity, and synthetic analogs have been developed.
Furthermore, HATs are also participating in ε-NH2 (Lys) acetylation of a variety of protein substrates, including, among others, the tumor suppressor p53 (Tang Y, Luo J, Zhang W, Gu W. Mol Cell (2006) 24:827-839, and other non-histone proteins (reviewed in Yang, X. J. The diverse superfamily of lysine acetyltransferases and their roles in leukemia and other diseases, Nucleic Acids Res 2004; 32:959-76). It has been proposed to rename this growing family of ε-NH2 (Lys) acetylating enzymes as KATs thus including all HATs (Allis, C. D., et al, 2007 Cell 131:633-636).
Despite the strong inhibitory activity of the CoA-type inhibitors against histone acetytransferase activity in isolated nuclei or permeabilized cells, or against purified p300/CBP HAT, they exhibit little growth inhibitory activity when used against whole cells, suggesting poor cellular uptake. However, the present inventors have discovered that these inhibitors penetrate the cell and affect a broader range of HAT activities without significantly affecting cell viability. Specific embodiments of the present invention include the multisubstrate histone acetyltransferase inhibitors, Spd-CoA and Spm-CoA, including the spermidine series Spd-1a/1b, Spd-2a/2b, Spd-3a/3b, Spd-4a/4b (in which the primary amino groups at the N1 or N8 atom of Spd are attached to the full or truncated CoA moiety), as well as the spermine series Spm-1, Spm-2, Spm-3, Spm-4 (in which the one of the primary amino groups of Spm is linked to the full or truncated CoA moiety). The present invention shows that these inhibitors are efficiently internalized into whole cells without permeabilization.
Without being bound by theory or mechanism, although inhibitor treatment has little effect on cell viability by itself, it has multiple effects on acetylation-dependent chromatin-associated functions in whole cells and sensitizes tumor cells to a variety of DNA-targeted treatments, including several commonly used chemotherapeutic agents. The inhibitors can also suppress uptake of natural polyamines needed for cell replication and can suppress cancer cell growth by suppressing intracellular polyamine accumulation as well.
The present invention has considerable clinical potential, particularly as a sensitizer to standard chemotherapy and radiation therapy, and as an inhibitor of polyamine metabolism by directly acting on the SSAT enzymes or by synergizing with inhibitors of the polyamine biosynthesis such as DFMO.